Covalent lipid modifications anchor heterotrimeric G proteins and Ras to the plasma membrane where they convey signals from cell surface receptors to intracellular effectors. The attachment of lipids is essential for their function. Palmitoylation is a reversible and regulated lipid modification. The goal of this project is to understand how palmitoylation of signaling proteins is regulated. The applicant has extensively characterized a palmitoyltransferase activity that modifies G-protein ? subunits and Ras. However, the molecular identity of that activity is unknown. A recent breakthrough has provided the tools to discover the molecular basis of palmitoyltransferase activities that modify signaling proteins.ERF2 and ERF4 (effect on Ras function) encode genes that are required for efficient Ras palmitoylation and plasma membrane association in Saccharomyces cerevisiae. Using assays developed by the applicant, a complex of Erf2p and Erf4p was shown to have palmitoyltransferase activity in vitro. Erf2p contains a DHHC-cysteine-rich domain (DHHC-CRD) that is essential for Ras palmitoylation in vitro and in vivo. This domain is found in six related proteins in S. cerevisiae. The applicant proposes to test the hypothesis that the DHHC-CRD domain defines a family of palmitoyltransferases that act on substrates in addition to Ras. We will determine whether proteins with DHHCCRD domains palmitoylate heterotrimeric G proteins in yeast. First, G-protein palmitoylation and function will be assayed in strains where DHHC-CRD genes have been inactivated to identify family members that contribute to Gprotein palmitoylation in vivo. Second, DHHC-CRD proteins will be assayed in vitro for palmitoyltransferase activity. Third, the subcellular localizatiOn of DHHC-CRD family members will be determined using fluorescence microscopy and subcellular fractionation. Localization is an important predictor of the pathways in which DHHCCRD proteins may function. Finally, the Erf2p/Erf4p complex will be purified and characterized biochemically to elucidate the mechanism of palmitate addition. Given the conservation of DHHC-CRD domains across species, the results of these studies in S. cerevisiae will have important implications for Ras and G-protein signaling in mammals.